注射用九种水溶性维生素冻干粉针的制备及其含量测定
摘要
本文将九种水溶性维生素:维生素B_1(VB_1)、维生素B_6(VB_6)、维生素B_(12)(VB_(12))、维生素C(VC)、烟酰胺、叶酸、D-生物素、核黄素磷酸钠、泛酸钠应用冷冻干燥法制备注射用九种水溶性维生素冻干粉针,优化了其处方和制备工艺,建立了含量分析方法。
第一部分考察了不同处方组成如冻干保护剂种类、药物溶液的粘度等,对冻干工艺及其冻结方式等对冻干产品的成形、水分含量及复溶速度等的影响。结果表明,选用海藻糖(300 mg/瓶)作为冻干保护剂,4 mL灌装量,反复预冻的方法进行制备注射用九种水溶性维生素冻干粉针效果较好。
第二部分应用高效液相色谱法(HPLC)对九种维生素成分进行含量分析检测,实验分别分为三组进行测定:
(1)叶酸、D-生物素、对羟基苯甲酸甲酯的含量测定
采用Diamonsil~(TM)ODS色谱柱,磷酸二氢钾缓冲液.乙腈为流动相,进行梯度洗脱,流速为1.5 mL/min,柱温30℃,检测波长200 nm。在该色谱条件下,叶酸、D-生物素、对羟基苯甲酸甲酯线性范围分别在8.05～24.15μg/mL(R=0.9995)、1.27～3.81μg/mL(R=0.9992)、9.95～29.85μg/mL(R=0.9991),三种成份回收率为96.3～104.2%,日间和日内精密度的RSD都在3.60%以内,最低检测浓度分别为0.30μg/mL、1.00μg/mL、0.10μg/mL。
(2)烟酰胺、VB_6、VB_1、泛酸钠、VC、核黄素磷酸钠的含量测定
采用DIKMA氨基色谱柱,0.02 mol/L磷酸二氢钾溶液-乙腈(27:73,V/V)为流动相,流速为1.0 mL/min,柱温30℃:烟酰胺、VB_1、VB_6、泛酸钠、VC的紫外检测波长为214nm;核黄素磷酸钠荧光检测波长λ_(ex)=445nm,λ_(em)=520nm。在该色谱条件下,烟酰胺、VB_6、VB_1、泛酸钠、VC、核黄素磷酸钠线性范围分别30.22～90.66μg/mL(R=0.9999)、3.76～11.28μg/mL(R=0.9992)、2.38～7.14μg/mL(R=0.9999)、12.44～37.32μg/mL(R=0.9995)、85.06～255.18μg/mL(R=0.9998)、1.94～11.64μg/mL(R=0.9999)。六种成份回收率为96.8～104.9%,日间和日内精密度的RSD都在3.40%以内,最低检测浓度分别为0.15、0.30、0.80、5.00、1.40、0.15μg/mL。
(3)VB_(12)的含量测定
采用Diamonsil~(TM)ODS色谱柱,以磷酸盐缓冲溶液-乙腈(90∶10,V/V)作为A流动相,乙腈-水-磷酸(499∶499∶2,V/V/V)作为B流动相,进行梯度洗脱,柱温30℃,检测波长200nm,在该色谱条件下,VB_(12)线性范围为0.5～1.5μg/mL(R=0.9996),回收率101.0～102.5%,日间和日内精密度的RSD都在1.80%以内,最低检测浓度分别为0.40μg/mL。
第三部分采用高效毛细管电泳法(HPCE)的胶束毛细管色谱(MECC)模式分离和同时测定注射用水溶性维生素的含量。应用未涂层熔融石英毛细管67cm(有效长度55cm)×50μm;运行缓冲液为50 mmol/L SDS-50 mmol/L的硼酸钠溶液(pH 8.33);6.89 KPa×10 sec压力进样;运行电压15.0 kV;检测波长214 nm;毛细管柱温25℃。在该色谱条件下,注射用水溶性维生素中的九种成份分离完全,VB_1、VB_6、VB__(12)、VC、烟酰胺、叶酸、D-生物素、核黄素磷酸钠、泛酸钠范线性范围分别为2.405～19.24μg/mL(R=0.9998)、3.80～30.40 gg/mL(R=0.9997)、0.39～3.12μg/mL(R=0.9992)、87.50～700.00 gg/mL(R=0.9990)、31.00～248.00μg/mL(R=0.9991)、0.31～2.48μg/mL(R=0.9990)、0.465～3.72μg/mL(R=0.9993)、3.80～30.40μg/mL(R=0.9982)、4.125～33.00μg/mL(R=0.9995):九种成份回收率为95.8～100.8%;日间和日内精密度峰面积的RSD在3.30%以内,迁移时间的RSD在2.70%以内;最低检测浓度分别为0.18、0.16、0.10、0.45、0.60、0.10、0.12、0.23、0.41μg/mL,与高效液相色谱分析方法相比较,此方法更加方便、简单。
本论文针对冷冻干燥法制备注射用九种水溶性维生素冻干粉针,应用高效液相色谱法和高效毛细管电泳法进行含量分析,并且对研究中出现的问题进行了改进,为注射用九种水溶性维生素冻干粉针的开发提供了实验依据。
In this paper,the lyophillization of nine water-soluble vitamins for injection was investigated,which included vitamin B_1(VB_1),vitamin B_6(VB_6),vitamin B_(12)(VB_(12)), vitamin C(VC),Nicotinamide,folic acid,D-Biotin,riboflavin 5'-phosphate sodium, D-pantothenic acid sodium salt.Content determination of nine water-soluble vitamins for injection was observed.
In the first chapter,we had a research for the influencing factors such as the excipient,viscosity of solution and lyophillization manner on the formula and preparation technology of the lyophilized nine water-soluble vitamins.The results indicted that trehalose(300 mg/ampoule)as supporting agent,4 mL of drug solution in an ampoule and pre-freeze in a repeated way were chosen to make the lyophillization product.
In the second chapter,nine vitmins were determined by high performance liquid chromatography(HPLC).The work includes three parts.
(1)Simultaneous determination of biotin,folic acid and methyl p-hydroxybenzoate in water-soluble vitamin for injection by HPLC
The HPLC system equipped with a 5μm ODS C_(18)250×4.6 mm column using gradient elution program was adopted.The mobile phase was potassium dihydrogen phosphate buffer - acetonitrile,and the column temperature was 30℃.The flow rate was 1.5 mL/min,and the detection wavelength was 200 nm.The linear ranges of biotin,folic acid and methyl p-hydroxybenzoate were 8.05～24.15μg/mL,1.27～3.81μg/mL,9.95～29.85μg/mL,respectively.In the linear range the correlation coefficients were 0.9991～0.9995.The average recoveries ranged from 96.3%to 104.2%with less than 3.60%of intra-day and inter-day RSD.The detection limits were 0.30μg/mL,1.00μg/mL,0.10μg/mL,respectively
(2)Determination of nicotinamide,VB_6,VB_1,D-pantothenic acid sodium salt,VC, riboflavin 5'-phosphate sodium in water-soluble vitamin for Injection by HPLC
The HPLC system equipped with a 5μm NH_2 250×4.6 mm column was adopted. The mobile phase was 0.02 mol/L potassium dihydrogen phosphate buffer-acetonitrile (27:73,V/V),and the column temperature was 30℃.The flow rate was 1.0 mL/min. To simultaneously analyze nicotinamide,VB_6,VB_1,D-pantothenic acid sodium salt, VC,the UV detection wavelength was set at 214 ran.Riboflavin 5'-phosphate sodium was investigated by fluorescence detection.Theλ_(ex)was 445 nm,andλ_(ex)was 520 nm. The linear ranges of nicotinamide,VB_6,VB_1,D-pantothenic acid sodium salt,VC, riboflavin 5'-phosphate sodium were 30.22～90.66μg/mL,3.76～11.28μg/mL, 2.38～7.14μg/mL,12.44～37.32μg/mL,85.06～255.18～g/mL,1.94～11.64μg/m,,respectively.In the linear range the correlation coefficients ranged from 0.9992 to 0.9999.The average recoveries were 96.3%-104.2%with less than 3.40%of intra-day and inter-day RSD.The detection limits were 0.15,0.30,0.80,5.00,1.40, 0.15μg/mL,respectively.
(3)Determination of VB 12 in water-soluble vitamin for injection by HPLC
The HPLC system was adopted,on a 5μm ODS C_(18)250×4.6 mm column, gradient elution,using the mobile phase of A:phosphate buffer-acetonitrile(90:10, V/V),and B:acetonitrile-water-phosphate acid(499:499:2,V/V/V).The column temperature was 30℃.The detection wavelength was 200 nm.The linear range of VB_(12)was 0.50～1.50μg/mL.In the linear range the correlation coefficients was 0.9996.The average recoveries ranged from 101.0%to 102.5%.The intra-day and inter-day RSD was less than 1.80%.The detection limit was 0.40μg/mL.
In the third chapter,nine water-soluble vitamin for injection were simultaneously determined by high performance capillary electrophoresis(HPCE).Micellar electrokinetic capillary chromatography(MEKC)modes was operated.The system was equipped with fused-silica capillary column:67 cm(efficiency length 55 cm)×50μm.The running buffer was 50 mmol/L SDS-50 mmol/L Na_2B_4O_7·10H_2O solution (pH = 8.33).The eletroinjection was 6.89 KPa×l0 sec with 15 kv running voltage. The detection wavelength was 214 nm.The column temperature was 25℃.The linear ranges of VB_1,VB_6,VB_(12),VC,nicotinamide,folic acid,D-biotin,riboflavin 5'-phosphate sodium,D-pantothenic acid sodium salt were 2.405～19.24μg/mL, 3.8～30.4μg/mL,0.39～3.12μg/mL,87.5～700.0μg/mL,31～248μg/mL,0.31～2.48μg/mL,0.465～3.72μg/mL,3.8～30.4μg/mL,4.125～33.0μg/mL,respectively. In the linear range the correlation coefficients were 0.9982 - 0.9998.The average recoveries ranged from 95.8%to 100.8%.The mean peak area and migration time of intra-day and inter-day RSD was less than 3.30%.The detection limits were 0.18μg/mL,0.16μg/mL,0.10μg/mL,0.45μg/mL,0.60μg/mL,0.10μg/mL,0.12μg/mL, 0.23μg/mL;0.41μg/mL,respectively.Compared with the HPLC method,HPCE was more convenient.
In general,the nine water-soluble vitamins for injection were prepared by lyophillization and determined by HPLC and HPCE.This work made a good foundation of the development of this preparation.
第一部分考察了不同处方组成如冻干保护剂种类、药物溶液的粘度等,对冻干工艺及其冻结方式等对冻干产品的成形、水分含量及复溶速度等的影响。结果表明,选用海藻糖(300 mg/瓶)作为冻干保护剂,4 mL灌装量,反复预冻的方法进行制备注射用九种水溶性维生素冻干粉针效果较好。
第二部分应用高效液相色谱法(HPLC)对九种维生素成分进行含量分析检测,实验分别分为三组进行测定:
(1)叶酸、D-生物素、对羟基苯甲酸甲酯的含量测定
采用Diamonsil~(TM)ODS色谱柱,磷酸二氢钾缓冲液.乙腈为流动相,进行梯度洗脱,流速为1.5 mL/min,柱温30℃,检测波长200 nm。在该色谱条件下,叶酸、D-生物素、对羟基苯甲酸甲酯线性范围分别在8.05～24.15μg/mL(R=0.9995)、1.27～3.81μg/mL(R=0.9992)、9.95～29.85μg/mL(R=0.9991),三种成份回收率为96.3～104.2%,日间和日内精密度的RSD都在3.60%以内,最低检测浓度分别为0.30μg/mL、1.00μg/mL、0.10μg/mL。
(2)烟酰胺、VB_6、VB_1、泛酸钠、VC、核黄素磷酸钠的含量测定
采用DIKMA氨基色谱柱,0.02 mol/L磷酸二氢钾溶液-乙腈(27:73,V/V)为流动相,流速为1.0 mL/min,柱温30℃:烟酰胺、VB_1、VB_6、泛酸钠、VC的紫外检测波长为214nm;核黄素磷酸钠荧光检测波长λ_(ex)=445nm,λ_(em)=520nm。在该色谱条件下,烟酰胺、VB_6、VB_1、泛酸钠、VC、核黄素磷酸钠线性范围分别30.22～90.66μg/mL(R=0.9999)、3.76～11.28μg/mL(R=0.9992)、2.38～7.14μg/mL(R=0.9999)、12.44～37.32μg/mL(R=0.9995)、85.06～255.18μg/mL(R=0.9998)、1.94～11.64μg/mL(R=0.9999)。六种成份回收率为96.8～104.9%,日间和日内精密度的RSD都在3.40%以内,最低检测浓度分别为0.15、0.30、0.80、5.00、1.40、0.15μg/mL。
(3)VB_(12)的含量测定
采用Diamonsil~(TM)ODS色谱柱,以磷酸盐缓冲溶液-乙腈(90∶10,V/V)作为A流动相,乙腈-水-磷酸(499∶499∶2,V/V/V)作为B流动相,进行梯度洗脱,柱温30℃,检测波长200nm,在该色谱条件下,VB_(12)线性范围为0.5～1.5μg/mL(R=0.9996),回收率101.0～102.5%,日间和日内精密度的RSD都在1.80%以内,最低检测浓度分别为0.40μg/mL。
第三部分采用高效毛细管电泳法(HPCE)的胶束毛细管色谱(MECC)模式分离和同时测定注射用水溶性维生素的含量。应用未涂层熔融石英毛细管67cm(有效长度55cm)×50μm;运行缓冲液为50 mmol/L SDS-50 mmol/L的硼酸钠溶液(pH 8.33);6.89 KPa×10 sec压力进样;运行电压15.0 kV;检测波长214 nm;毛细管柱温25℃。在该色谱条件下,注射用水溶性维生素中的九种成份分离完全,VB_1、VB_6、VB__(12)、VC、烟酰胺、叶酸、D-生物素、核黄素磷酸钠、泛酸钠范线性范围分别为2.405～19.24μg/mL(R=0.9998)、3.80～30.40 gg/mL(R=0.9997)、0.39～3.12μg/mL(R=0.9992)、87.50～700.00 gg/mL(R=0.9990)、31.00～248.00μg/mL(R=0.9991)、0.31～2.48μg/mL(R=0.9990)、0.465～3.72μg/mL(R=0.9993)、3.80～30.40μg/mL(R=0.9982)、4.125～33.00μg/mL(R=0.9995):九种成份回收率为95.8～100.8%;日间和日内精密度峰面积的RSD在3.30%以内,迁移时间的RSD在2.70%以内;最低检测浓度分别为0.18、0.16、0.10、0.45、0.60、0.10、0.12、0.23、0.41μg/mL,与高效液相色谱分析方法相比较,此方法更加方便、简单。
本论文针对冷冻干燥法制备注射用九种水溶性维生素冻干粉针,应用高效液相色谱法和高效毛细管电泳法进行含量分析,并且对研究中出现的问题进行了改进,为注射用九种水溶性维生素冻干粉针的开发提供了实验依据。
In this paper,the lyophillization of nine water-soluble vitamins for injection was investigated,which included vitamin B_1(VB_1),vitamin B_6(VB_6),vitamin B_(12)(VB_(12)), vitamin C(VC),Nicotinamide,folic acid,D-Biotin,riboflavin 5'-phosphate sodium, D-pantothenic acid sodium salt.Content determination of nine water-soluble vitamins for injection was observed.
In the first chapter,we had a research for the influencing factors such as the excipient,viscosity of solution and lyophillization manner on the formula and preparation technology of the lyophilized nine water-soluble vitamins.The results indicted that trehalose(300 mg/ampoule)as supporting agent,4 mL of drug solution in an ampoule and pre-freeze in a repeated way were chosen to make the lyophillization product.
In the second chapter,nine vitmins were determined by high performance liquid chromatography(HPLC).The work includes three parts.
(1)Simultaneous determination of biotin,folic acid and methyl p-hydroxybenzoate in water-soluble vitamin for injection by HPLC
The HPLC system equipped with a 5μm ODS C_(18)250×4.6 mm column using gradient elution program was adopted.The mobile phase was potassium dihydrogen phosphate buffer - acetonitrile,and the column temperature was 30℃.The flow rate was 1.5 mL/min,and the detection wavelength was 200 nm.The linear ranges of biotin,folic acid and methyl p-hydroxybenzoate were 8.05～24.15μg/mL,1.27～3.81μg/mL,9.95～29.85μg/mL,respectively.In the linear range the correlation coefficients were 0.9991～0.9995.The average recoveries ranged from 96.3%to 104.2%with less than 3.60%of intra-day and inter-day RSD.The detection limits were 0.30μg/mL,1.00μg/mL,0.10μg/mL,respectively
(2)Determination of nicotinamide,VB_6,VB_1,D-pantothenic acid sodium salt,VC, riboflavin 5'-phosphate sodium in water-soluble vitamin for Injection by HPLC
The HPLC system equipped with a 5μm NH_2 250×4.6 mm column was adopted. The mobile phase was 0.02 mol/L potassium dihydrogen phosphate buffer-acetonitrile (27:73,V/V),and the column temperature was 30℃.The flow rate was 1.0 mL/min. To simultaneously analyze nicotinamide,VB_6,VB_1,D-pantothenic acid sodium salt, VC,the UV detection wavelength was set at 214 ran.Riboflavin 5'-phosphate sodium was investigated by fluorescence detection.Theλ_(ex)was 445 nm,andλ_(ex)was 520 nm. The linear ranges of nicotinamide,VB_6,VB_1,D-pantothenic acid sodium salt,VC, riboflavin 5'-phosphate sodium were 30.22～90.66μg/mL,3.76～11.28μg/mL, 2.38～7.14μg/mL,12.44～37.32μg/mL,85.06～255.18～g/mL,1.94～11.64μg/m,,respectively.In the linear range the correlation coefficients ranged from 0.9992 to 0.9999.The average recoveries were 96.3%-104.2%with less than 3.40%of intra-day and inter-day RSD.The detection limits were 0.15,0.30,0.80,5.00,1.40, 0.15μg/mL,respectively.
(3)Determination of VB 12 in water-soluble vitamin for injection by HPLC
The HPLC system was adopted,on a 5μm ODS C_(18)250×4.6 mm column, gradient elution,using the mobile phase of A:phosphate buffer-acetonitrile(90:10, V/V),and B:acetonitrile-water-phosphate acid(499:499:2,V/V/V).The column temperature was 30℃.The detection wavelength was 200 nm.The linear range of VB_(12)was 0.50～1.50μg/mL.In the linear range the correlation coefficients was 0.9996.The average recoveries ranged from 101.0%to 102.5%.The intra-day and inter-day RSD was less than 1.80%.The detection limit was 0.40μg/mL.
In the third chapter,nine water-soluble vitamin for injection were simultaneously determined by high performance capillary electrophoresis(HPCE).Micellar electrokinetic capillary chromatography(MEKC)modes was operated.The system was equipped with fused-silica capillary column:67 cm(efficiency length 55 cm)×50μm.The running buffer was 50 mmol/L SDS-50 mmol/L Na_2B_4O_7·10H_2O solution (pH = 8.33).The eletroinjection was 6.89 KPa×l0 sec with 15 kv running voltage. The detection wavelength was 214 nm.The column temperature was 25℃.The linear ranges of VB_1,VB_6,VB_(12),VC,nicotinamide,folic acid,D-biotin,riboflavin 5'-phosphate sodium,D-pantothenic acid sodium salt were 2.405～19.24μg/mL, 3.8～30.4μg/mL,0.39～3.12μg/mL,87.5～700.0μg/mL,31～248μg/mL,0.31～2.48μg/mL,0.465～3.72μg/mL,3.8～30.4μg/mL,4.125～33.0μg/mL,respectively. In the linear range the correlation coefficients were 0.9982 - 0.9998.The average recoveries ranged from 95.8%to 100.8%.The mean peak area and migration time of intra-day and inter-day RSD was less than 3.30%.The detection limits were 0.18μg/mL,0.16μg/mL,0.10μg/mL,0.45μg/mL,0.60μg/mL,0.10μg/mL,0.12μg/mL, 0.23μg/mL;0.41μg/mL,respectively.Compared with the HPLC method,HPCE was more convenient.
In general,the nine water-soluble vitamins for injection were prepared by lyophillization and determined by HPLC and HPCE.This work made a good foundation of the development of this preparation.
引文
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